2011-2012 Catalogue - PeproTech, Inc.

PEPROTECH ®www.peprotech.comWestern Transfer Troubleshooting GuideProblem Possible source SuggestionWeak/No Signal Insufficient amount of protein • Increase amount of protein in sample.present• Confirm protein is present through an alternateˇmethod.ˇ Gel ran too long • Decrease running time and monitor the gel byˇwatching dye front and ensuring the current isˇturned off when dye front is near bottom of gel.ˇ Old or degraded protein samples • Test a fresh sample of target protein.ˇ Protein over-transferred/ • Use a membrane with a smaller pore size.ˇ did not transfer correctly • Decrease voltage for lower MW proteins (lessˇthan 10kDa).ˇ• Make sure membrane is pre-soaked accordingˇto manufacturer’s instructions.ˇ• Check and optimize transfer time, asˇappropriate transfer time will vary with MW ofˇthe target protein.ˇ• Make sure transfer “sandwich” is set up correctlyˇand there is sufficient contact between gel andˇmembrane. (Please see our Western Transferˇprotocol for correct transfer “sandwich” set up.)ˇ• Ensure the correct transfer buffer is being used.ˇ• The presence of SDS during transfer can reduceˇthe binding of the protein when using aˇnitrocellulose membrane.ˇ• Add 20% methanol to transfer buffer to increaseˇbinding.ˇ• Check protein transfer by staining the membraneˇwith a reversible stain, such as Ponceau S, toˇvisualize major bands.ˇ Protein has a pI greater than 9 • Substitute buffer with a higher pH buffer (i.e.ˇ CAPS buffer, pH 10.5).ˇ Protein masked by blocking buffer • Test using a different blocking buffer.ˇ• Optimize concentration of protein in blockingˇbuffer. We recommend 3% Non-Fat Dry Milk inˇ diH 2O.ˇ Enzyme inhibitor present (i.e. azide) • Make sure buffers do not contain sodium azide,ˇas this will interfere with the HRP signal.ˇ Old or degraded primary antibody • Check manufacturer’s recommendations forˇstorage and/or expiration date. If expired or pastˇstorage time, purchase new antibody.ˇ• If antibody was subjected to repeated freeze/thawˇcycles purchase new antibody, as this can affectˇthe structure and protein:antibody binding.ˇ• Perform a Dot Blot to ensure antibody is still active.ˇ Insufficient amount of primary • Check manufacturer’s recommendation forˇ and/or secondary antibody antibody concentration.ˇ• Test a range of concentrations to find the optimalˇcondition for individual assay.ˇ Incorrect primary antibody • Ensure primary antibody reacts with the targetˇprotein from the species being studied (i.e.ˇ Rabbit-anti-human VEGF Human VEGF).INFORMATION & TECHNICAL SUPPORTBestellungen: Deutschland: Tel: 0800 436 99 10 • email: info@peprotech.dePour commander: France: Tel: +33 (0)1 46 24 58 20 • email: info@peprotechfr.comTo Order: NORDIC: Tel: +46 (0)8 640 41 07 • email: info@peprotech.se167